The present invention relates to mass spectroscopy systems, and more particularly, but without limitation, relates to an apparatus and method for calibrating a mass spectrometer by internally introducing calibration masses at a post-source stage of the mass spectrometer.
For many years, mass spectrometers have proved to be a valuable tool for analyzing the chemical composition of complex mixtures of substances. Constituent molecules are ionized and then differentiated according to the ratio of their mass to their ionization charge (m/z). In recent times, numerous improvements have been made in sample preparation and ionization techniques, which collectively pertain to the xe2x80x9cion sourcexe2x80x9d region of the mass spectrometer. Atmospheric Pressure Ionization (API) techniques, such as Electrospray (ESI), Atmospheric Pressure Chemical Ionization (APCI), Atmospheric Pressure Photoionization (APPI) and Atmospheric Pressure Matrix Assisted Laser Desorption/Ionization (AP MALDI) are now commonly used to generate analyte ions from fluid samples. These techniques have improved the sensitivity of mass spectrometer systems by increasing the concentration of ionized analyte molecules that enter the mass spectrometer and reach detectors downstream.
In Electrospray sources, an analyte solution from a source apparatus, such as a liquid chromatography column, is ejected from a needle as a liquid stream. Instabilities in the liquid stream generated by nebulizing means such as a nebulizing gas, pneumatic assist and/or ultrasonic waves result in breakup of the stream into droplets, many of which bear electric charge as a result of the needle being at high potential with respect to surrounding conductors, or due to triboelectric effects. The charged droplets are desolvated by evaporation, freeing desolvated, ionized analyte molecules. The analyte ions are then directed into a mass spectrometer interface from which the constituent molecules are transported through one or more vacuum stages downstream to a mass analyzer. At the mass analyzer the analyte ions are filtered and then detected.
Concurrent improvements in mass analysis techniques, such as Time-Of-Flight (TOF) and Magnetic Sector and Fourier Transform Ion Cyclotron Resonance (FTICR), have made mass assignment accuracies on the order of 1 to 10 ppm (parts per million) feasible. However, this level of accuracy requires a level of instrument stability and repeatability that is not always attainable due to xe2x80x9cdriftxe2x80x9d caused by fluctuations in ambient temperature, spectrometer chamber pressures, and applied voltages. To adjust to such drift, instruments are calibrated using masses that are known, using a process referred to as mass calibration. According to this technique, known compounds, herein referred to as lock masses, having characteristic m/z ratios, are typically analyzed either in conjunction or sequentially with samples of unknown compounds (analytes). The resulting mass spectrum contains one or more internal calibration peaks corresponding to the m/z ratio of the lock masses which can then serve as a scale by which the masses of peaks corresponding to the unknown compounds can be measured. Methods for use of lock masses in calibration of analyte mass spectra are well known in the art.
In one conventional method of mass calibration, lock masses are mixed with the unknown sample in solution prior to ionization in the ion source. This conventional method suffers from the problem of contamination as the lock masses contaminate transfer lines and capillary tips, and also suppress the ionization efficiency of the sample compounds during the ionization process. At the high accuracy threshold required for distinguishing between large molecular-weight compounds, even slight instrument drift can alter analysis results, so that it is advantageous to run successive analyses at a high-throughput rate before large drift fluctuations materialize. At such high-throughput rates, lock mass contamination becomes a more important issue because the residue of the lock mass left over from previous analysis runs may be difficult to eliminate before succeeding analysis runs take place.
Recently, techniques have been developed for introducing lock masses externally from the sample, which purport to reduce the effects of contamination. In xe2x80x9cMultiple Sample Introduction Mass Spectroscopy,xe2x80x9d U.S. Pat. No. 6,207,954, separate API source probes introduce two or more compounds including a lock mass into the ion source chamber simultaneously. In xe2x80x9cMulti-inlet mass spectrometer,xe2x80x9d European Patent Application No. 0 966 022, multiple Electrospray probes aligned at different angles spray toward a spinning chamber that has an opening that aligns with a portion of the probes. The charged-particle jets emitted by the portion of probes that are aligned with the opening enter the sampling orifice of the mass spectrometer. In each of these external introduction techniques, the analyte sample and the lock mass ions can be emitted from separate probes, reducing interaction between the lock mass and sample in solution and probe contamination.
However, both techniques require duplication of sample probes and injectors, a complex ion source interface, and both are adapted specifically for Electrospray ionization sources. Additionally, because the lock mass molecules are introduced within the ion source, some remnant level of contamination of the ion source and/or mass spectrometer interface is unavoidable. It would therefore be advantageous to provide a simplified lock mass introduction technique that does not depend on the ion source implementation and does not cause any source/interface contamination.
Furthermore, in the field of tandem mass spectroscopy (MS/MS) where the second MS stage is capable of exact mass determination, there is added complication with respect to the addition of lock masses. MS/MS involves selection of a narrow range of xe2x80x9cparentxe2x80x9d ions with a first mass analyzer or mass filter stage, fragmentation of the parent ions in a collision chamber, creating xe2x80x9cdaughter ionsxe2x80x9d, and then analysis of the composition of the daughter ions in a second mass analyzer. In this, arrangement, a lock mass introduced at the ion source must pass through both the first mass analyzer and the collision cell, which requires that the lock mass and its daughter ions be in the same mass range as the parent ion of interest because they would otherwise be filtered and/or fragmented away. Therefore, the current method is to use the parent ion as the lock mass. This method requires that the parent ion be known, and also that the parent ion not be completely fragmented in the collision cell, since a portion must pass through to the second mass analyzer. These requirements decrease the number of daughter ions available and provide low ion statistics for both the parent and daughter ions. In addition, proper mass axis calibration requires the m/z ratio of the daughter ions to be within range of the parent ion. The number of lock masses available is thereby limited. It would accordingly be advantageous to provide a simple lock mass introduction technique for MS/MS that does not suffer from these constraints, and in particular, does not require use of the parent ion as the lock mass.
The present invention provides a mass calibration apparatus in which lock masses are internally introduced at a post-source stage of a mass spectrometer. Lock mass ions mix with the analyte ions in the ion optics prior to mass analysis.
In different embodiments, the source of lock mass ions may include various means for ionizing lock mass molecules including but not limited to photoionization, field desorption-ionization, electron ionization, and thermal ionization means.
The present invention also provides internal introduction of lock masses into a tandem mass spectrometer. The tandem mass spectrometer comprises a first mass analyzer, a collision cell and a second mass analyzer. The collision cell receives selected analyte ions from the first mass analyzer and includes collision gas that fragments the selected analyte ions into daughter ions. In some embodiments, the first mass analyzer and the collision cell are combined into a single unit that has the functions of both. Examples of these embodiments include use of a quadrupole ion trap or a linear ion trap. A lock mass source introduces lock mass molecules directly into the collision cell without subjecting the lock mass molecules to fragmentation by the collision gas, and a lock mass ionization unit ionizes the lock mass within the collision cell. In some embodiments, the lock mass introduction and ionization can be into ion optics located after the collision cell and before the second mass analyzer.
The present invention also provides a method for mass calibration of analyte ions with lock masses in a mass spectrometer having an analyte ion source, ion optics, and a mass analyzer, by creating lock mass ions within the ion optics. According to one embodiment, the step of creating lock mass ions comprises introducing lock mass molecules into the ion optics. According to a second embodiment, the step of creating lock mass ions comprises ionizing lock mass molecules within the ion optics. These steps are not exclusive and according to another embodiment lock mass ions are created by introducing lock mass molecules into the ion optics and ionizing the lock mass molecules introduced within the ion optics. In these methods, lock mass ions are ionized substantially in or near the downstream path of the analyte ions so that both analyte ions and lock mass ions thereafter travel along the same path downstream and are detected and analyzed together.
In addition, the present invention provides a method for mass calibration of a tandem mass spectrometer that includes a collision cell by creating lock mass ions within the collision cell. According to one embodiment, the step of creating lock mass ions comprises introducing lock mass molecules into the collision cell. According to a second embodiment, the step of creating lock mass ions comprises ionizing lock mass molecules within the collision cell. These steps are not exclusive and according to another embodiment, lock mass ions are created by introducing lock mass molecules into the collision cell and ionizing the lock mass molecules within the collision cell.
The present invention also provides a method for mass calibration of a tandem mass spectrometer that includes ion optics for transporting analyte daughter ions to a mass analyzer by creating lock mass ions within the ion optics. The lock mass ions are created by introducing lock mass molecules into the ion optics and/or ionizing lock mass molecules within the ion optics.
According to these methods for calibrating a tandem mass spectrometer, lock mass molecules are introduced and ionized in the path of analyte daughter ions. The lock mass ions are then guided and transported together with the analyte daughter ions for detection and analysis.